Sequence-specific transcription factors (TFs) regulate gene expression by binding to cis-regulatory elements in promoter and enhancer DNA. While studies of TF–DNA binding have focused on TFs' intrinsic preferences for primary nucleotide sequence motifs, recent studies have elucidated additional layers of complexity that modulate TF–DNA binding. In this review, we discuss technological developments for identifying TF binding preferences and highlight recent discoveries that elaborate how TF interactions, local DNA structure, and genomic features influence TF–DNA binding. We highlight novel approaches for characterizing functional binding site motifs that promise to inform our understanding of how TF binding controls gene expression and ultimately contributes to phenotype.

Small, noncoding RNAs (sncRNAs), including microRNAs (miRNAs), impact diverse biological events through the control of gene expression and genome stability. However, the role of these sncRNAs in aging remains largely unknown. To understand the contribution of sncRNAs to the aging process, we performed small RNA profiling by deep-sequencing over the course of Caenorhabditis elegans (C. elegans) aging. Many small RNAs, including a significant number of miRNAs, change their expression during aging in C. elegans. Further studies of miRNA expression changes under conditions that modify lifespan demonstrate the tight control of their expression during aging. Adult-specific loss of argonaute-like gene-1 (alg-1) activity, which is necessary for miRNA maturation and function, resulted in an abnormal lifespan, suggesting that miRNAs are, indeed, required in adulthood for normal aging. miRNA target prediction algorithms combined with transcriptome data and pathway enrichment analysis revealed likely targets of these age-associated miRNAs with known roles in aging, such as mitochondrial metabolism. Furthermore, a computational analysis of our deep-sequencing data identified additional age-associated sncRNAs, including miRNA star strands, novel miRNA candidates, and endo-siRNA sequences. We also show an increase of specific transfer RNA (tRNA) fragments during aging, which are known to be induced in response to stress in several organisms. This study suggests that sncRNAs including miRNAs contribute to lifespan regulation in C. elegans, and indicates new connections between aging, stress responses, and the small RNA world.

Sequencing of exomes and genomes has revealed abundant genetic variation affecting the coding sequences of human transcription factors (TFs), but the consequences of such variation remain largely unexplored. We developed a computational, structure-based approach to evaluate TF variants for their impact on DNA-binding activity and used universal protein binding microarrays to assay sequence-specific DNA-binding activity across 41 reference and 117 variant alleles found in individuals of diverse ancestries and families with Mendelian diseases. We found 77 variants in 28 genes that affect DNA-binding affinity or specificity and identified thousands of rare alleles likely to alter the DNA-binding activity of human sequence-specific TFs. Our results suggest that most individuals have unique repertoires of TF DNA-binding activities, which may contribute to phenotypic variation.